The present invention is concerned with mutant forms of creatine amidinohydrolase which are more stable than the wild type enzyme and are, therefore, more suitable for the enzymatic determination of the creatinine content of a body fluid, such as serum, plasma and urine.
The enzyme creatine amidinohydrolase (EC 3.5.3.3) is used industrially for the determination of creatinine. It is used, inter alia, in clinical analysis for the diagnosis of kidney diseases in which the creatinine content occurring in the serum and urine differ from those of the healthy organism. Micro-organisms are known, for example types of Pseudomonas, which, with induction by creatine, are able to produce creatine amidinohydrolase in sufficient amount so that work up is worthwhile; however the achievable yields and the costs of isolation of the enzyme are still limiting factors for the industrial use of the enzyme.
As is described in Federal Republic of Germany Patent Specification No. 35 00 184, it is possible to isolate from Pseudomonas putida the gene coding creatine amidohydrolase and to introduce it, for example, into plasmid pBR 322. After transformation of a micro-organism of the genus Escherichia coli or Pseudomonas putida with the new plasmid, it is possible to obtain creatine amidinohydrolase constitutively therefrom. This genetechnological production of creatine amidinohydrolase is more effective and easier to carry out than via isolation of the enzyme from an induced micro-organism which does not contain a plasmid. However, this process has the disadvantage that the wild type enzyme obtained therefrom has only a limited detergent and thermal stability and thus is not optimally suitable for use in clinical test processes.
It is an object of the present invention to provide creatine amidinohydrolase mutants which do not display the described disadvantages of the prior art or do so only to a slight extent.